1. Field of the Invention
This invention relates to a hybrid, which has a sugar having a reducing terminal and containing no carboxyl group bound with lysozyme through a peptide linkage and a process for producing the same, and the object is to enhance the stability of lysozyme. Further, another object is to provide an activated sugar which is a sugar having a reducing terminal and containing no carboxyl group bound with N-hydroxysuccinimide through a peptide linkage which activated sugar is necessary to produce the above sugar-lysozyme hybrid, and a process for producing the activated sugar.
2. Description of Related Art
Proteins are constituted by a primary structure formed by linking 20 kinds of amino acids and a steric structure defined by the above primary structure. It has already been known that proteins have various functions, and even proteins stable in vivo become unstable when used in vitro.
When superior functions of proteins are applied to various uses, proteins have the following drawbacks:
1 they are unstable to heat, alkalies and acids and liable to be denatured;
2 they are insoluble in organic solvents and liable to lose their activity;
3 they have antigenic properties; etc.
In order to overcome these drawbacks, proteins have been chemically modified. Such chemical modification of proteins into a protein hybrid has made it possible to compensate the above drawbacks. Various processes have been proposed for such chemical modification. Among these processes, the most often employed one is a process of using as a modifying agent, a polyethylene glycol (hereinafter abbreviated to PEG) which is a non-immunity synthetic high-molecular weight compound.
According to the above process, as illustrated below in the equations, a synthesized substance (an activated PEG) is prepared from monomethoxypolyethylene glycol and a cyanuric chloride (2,4,6-trichloro-S-triazine), followed by reacting this activated PEG with a protein to produce a PEG-protein hybrid.
Utilizing this protein-hybrid, various application examples, as shown below, have been reported, but practical commercially employed examples appear to be few. This is because the activated PEG is unstable; no product having uniform properties is obtained; cyanuric chloride raises a tohieity problem; the reaction of the activated PEG with proteins does not occur quantitatively and smoothly; etc. ##STR1##
The application examples of PEG-protein hybrid are shown below.
(1) PEG-asparaginase (T. Pharmac., Y. Kamisaki et al; Exp. Therap. 216, 410) .
This hybrid prolongs the half-life period in blood of asparaginase as an antitumor enzyme and reduces its antigenic properties.
(2) By utilizing a PEG-enzyme hybrid, enzyme reactions have been made possible even in organic solvents (Y. Imada et al; Trends in Biotechnology 4 190(1986), K. Takahashi et al; J.Org Chem 50 3414(1985) K. Takahashi et al; Enzyme 32 235 (1984) K.Takahashi et al; Bioch em Biophys Res. Commun:125 761(1984)).
Catalase, lipase, chymotrypsin, peroxidase, etc.
(3) PEG-adenosine deaminase (M.S. Hershfield et al; N. Engl. J. Mol. 316,493 (1985))
Among genetic enzyme-deficient phenomena, there is adenosine aminase (ADA)-deficient phenomenon. When this ADA is administered, if it is made up into PEG-ADA hybrid, it has been reported that this hybrid has effects of notably prolonging the half-life period in blood, etc.
(4) PEG-interleukin 2 (Taiji Imoto; Chemistry and Organism, Vol. 27, page 426, 1989)
Interleukin 2 which is a kind of lymphokines has been mass-produced according to recombinant DNA technique, but it is deficient in a sugar chain so that it is unstable, but when it is made up into PEG-interleukin 2 hybrid, it could have been stabilized and also its antitumor effect could have been improved.
In order to make up a hybrid with proteins, sugars have been also utilized besides PEG. As processes utilizing sugars, there are the following processes (i) to (iv), and the effects of the resulting hybrids are almost the same as those in the case of PEG: ##STR2##
The process (i) has drawbacks that the reaction with periodic acid is so severe that sugars are often decomposed; binding with proteins requires the use of a reducing agent; there is a possibility that proteins are denatured; etc.
The process (ii) has drawbacks that poisonous bromine cyanide is used; when proteins are bound, it is necessary to-severely adjust the pH in the process; etc.
The process (iii) has drawbacks that cyanuric chloride is poisonous; the reaction of cyanuric acid with sugars does not progress smoothly.
The process (iv) has drawbacks that sugars crosslink with each other due to epichlorohydrin; etc. The drawbacks common to these processes consist in that since the above respective substances react with --OH of the constituting sugars, the binding position is indefinite and also since they react with any --OH of sugars, the properties specific to the respective sugars are lost.
Lysozyme as a bacteriolytic enzyme has been broadly used for medical industry, etc., and in order to enhance its stability, high-level techniques are required or cumbersome operations are required; for example, the composition of constituting amino acids varies according to the gene engineering technique used; crosslinking is carried out between the objective amino acids (see, Taiji Imoto; Chemistry and Organism, Vol. 27, page 426, 1989).